The long-term goal of our laboratory is to understand how signal transduction by cell surface receptors is regulated. We would like to develop strategies for selectively activating or inhibiting these cellular activities and bypass limitations of the receptor (i.e. low receptor number or receptor desensitization). Extracellular ligands (growth factor, hormone, neurotransmitter, etc.) bind to unique cell surface receptors that induce intracellular, biochemical changes that are integrated to invoke specific changes in cell physiology. While the exquisite specificity of this system has been long appreciated, the molecular mechanism by which it occurs is poorly understood. Understanding how an overlapping set of biochemical responses produces a specific physiology is the key to this problem. To understand this process, we are using the prototypical receptor tyrosine kinase, the epidermal growth factor receptor (EGFR), as a model. The EGFR is critical for many developmental and homeostatic processes; stimulation of the EGFR leads to a variety of cellular changes including cell proliferation, differentiation, migration, and viability. Overexpression of the EGFR is associated with many cancers. The magnitude and duration of signaling to these biochemical intermediates dictates how cell physiology is altered. One way this occurs is through the internalization and degradation of the receptor following ligand binding. In addition to activating intracellular signaling pathways, ligand binding also causes most cells surface receptors to internalize via clathrin-coated pits. Once inside the cell, the ligand:receptor complex moves through a series of well-defined endocytic stages until it ultimately reaches the lysosome where it undergoes degradation. It has been shown previously that disrupting EGFR endocytosis can alter the response of the cell. However, these studies have been limited to distinguishing between cell surface and intracellular receptors. The overarching hypothesis of our research is that the endocytic pathway is a key positive and negative regulator of cell surface receptor signaling. In Aim 1, we will selectively disrupt EGFR trafficking through the endocytic pathway. We will assess EGFR signaling at several endocytic stages and determine whether differences in signaling occur due to changes in the receptor itself, receptor:effector interactions, or the duration/magnitude of signaling. In Aim 2 we will build on our recent findings that spatially restricting the EGFR in cancer cells (MDA-MB-468 cells) dramatically changes cell growth and viability properties. We will determine how the intracellular EGFRs produce apoptotic signals and whether changing the trafficking and signaling of EGFRs in other cell lines can cause them to undergo EGFR-mediated apoptosis. Finally, in Aim 3, we will explore how the endocytic pathway negatively regulates EGFR signaling. In these studies, we will determine the mechanism of signal inactivation and determine if altering the rate of inactivation is sufficient to change EGFR signaling. PUBLIC HEALTH RELEVANCE: The epidermal growth factor receptor (EGFR) is a fundamental cell surface protein that functions by detecting the presence of growth factors outside the cell and converting the information into biochemical changes within the cell. Proper function of the EGFR is necessary for development and homeostasis of the whole organism; overexpression/hyperactivation of the EGFR is associated with many cancers. The immediate goal of this research is to better understand the molecular mechanisms that regulate the biological and pathological functions of the EGFR and use that information for treatment of diseases associated with the EGFR.